Investigation of the enantioselectivity of acetylcholinesterase and butyrylcholinesterase upon inhibition by tacrine-iminosugar heterodimers

Abstract The copper-catalysed azide-alkyne cycloaddition was applied to prepare three enantiomeric pairs of heterodimers containing a tacrine residue and a 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) or 1,4-dideoxy-1,4-imino-L-arabinitol (LAB) moiety held together via linkers of variable lengths containing a 1,2,3-triazole ring and 3, 4, or 7 CH2 groups. The heterodimers were tested as inhibitors of butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE). The enantiomeric heterodimers with the longest linkers exhibited the highest inhibition potencies for AChE (IC50 = 9.7 nM and 11 nM) and BuChE (IC50 = 8.1 nM and 9.1 nM). AChE exhibited the highest enantioselectivity (ca. 4-fold). The enantiomeric pairs of the heterodimers were found to be inactive (GI50 > 100 µM), or to have weak antiproliferative properties (GI50 = 84–97 µM) against a panel of human cancer cells.


Introduction
Enzyme inhibition represents an attractive target for drug development 1 . Because enzymes are built up by chiral building blocks, amino acids, it is not surprising if only one member of an enantiomeric pair causes inhibition upon binding. Another alternative is that both enantiomers display various degrees of inhibition, due to different interaction modes [2][3][4] . One such example is the natural enantiomer 1a (Figure 1) of huperzine A, which is a 38-to 49-fold more potent AChE inhibitor than its unnatural enantiomer 1b 5, 6 . In fact, inhibition of cholinesterases (ChEs) is an attractive target for treatment of Alzheimer's disease (AD) and there are currently three ChE inhibitors on the list of FDA approved AD drugs 7 . (-)-Huperzine (1a) is not on the list of FDA approved drugs, but it was approved in China as a symptomatic AD drug 8,9 .The much stronger AChE inhibition exhibited by 1a compared to 1b, was partially rationalised by comparison of the X-ray structures of Torpedo californica acetylcholinesterase (TcAChE) complexed with enantiomers 1a and 1b, which demonstrated the presence and absence of an interaction between the ethylidene methyl of 1a and 1b, respectively, with His440 10 , which is a member of the catalytic triad almost on the bottom of a ca. 20 Å deep active gorge of TcAChE 11 .
(-)-Galantamine (2a) (Figure 1) is an FDA approved ChE inhibitor drug for the treatment of mild-to-moderate AD 7 . This alkaloid is a reversible AChE inhibitor and exhibits 53-times selectivity for AChE over BuChE 12 . X-ray studies of the TcAChE/(-)-galantamine complex revealed that the inhibitor binds in its acidic form at the base of the active gorge in the region between the acetyl hole and the catalytic anionic site (CAS) 13 . The protonated amine group is quite remote from Trp84 in CAS and thereby is not involved in any cation-p interactions with the Trp84 residue, which is in stark contrast to acetylcholine (ACh), whose quaternary ammonium group establishes a cation-p interaction with Trp84. Instead, the high affinity of (-)-galantamine for AChE was attributed to multiple moderate and weak interactions with the enzyme 13 . The inhibition of AChE by galantamine appears to be enantioselective, at 20 lM inhibitor concentration, as the natural enantiomer 2a (ca. 94% of inhibition) is a much stronger inhibitor than its unnatural antipode 2b (ca. 4% of inhibition) 14 , which indicates that several interactions with the enzyme are eliminated or attenuated when the configuration in all stereogenic centres of 2a is reversed.
Iminosugars are glycomimetics in which the ring oxygen atom has been replaced by a nitrogen atom 17 . Iminosugars are attractive as pharmaceutical candidates because they inhibit glycosidases without being metabolised by such enzymes 18 . Such properties have made iminosugars attractive as synthetic targets 19 and lead compounds for the treatment of various diseases such as viral infections, diabetes, type 2 diabetes, and lysosomal disorders 20 . In addition, it has been found that iminosugars are able to inhibit the growth of cancer cells 21,22 , without affecting the viability and mortality of normal cells 21 . To date, three iminosugars, namely, miglitol 23 , miglustat 24 , and migalastat 25 have been approved by FDA for the treatment of type 2 diabetes, Gaucher's disease, and Fabry's disease, respectively. Miglustat has also been found to reduce the production of amyloid b-peptide (Ab) 26 , which is a component of senile plaque in AD patients.
The ester group of ACh is held in place for hydrolysis by the catalytic triad in the active gorge of AChE by the aid of cation À p interactions with a Trp residue in CAS 27 . Because many iminosugars are protonated at physiological pH 28 , they were proposed to be capable of inhibiting ChEs 29 . Thereby, a series of iminosugars of various stereochemistry and substitution patterns have been tested as ChE inhibitors, displaying particularly good BuChE inhibition 29 . Following this line, some of us have reported the synthesis and ChE inhibitory testing of bivalent inhibitors in which a 1-deoxynojirimycin (1-DNJ) (4) binding unit is connected to a second binding unit, namely, aryl-substituted selenourea (exemplified by 5) 30 , catechol (exemplified by 6) 31 , tacrine (exemplified by 7) 32 , or benzotriazole (exemplified by 8) 33 binding unit ( Figure 2). Kinetic assays and modelling studies for the binding of 6, 7, and 8 to AChE indicated that they behave as dual binding site AChE inhibitors, which implies that they bind simultaneously to the peripheral anionic site (PAS) and CAS. A more surprising observation (given that the quaternary ammonium group of ACh participates in a cation-p interaction with a Trp residue in CAS) from the modelling studies was that when heterodimers 6 and 8 bind to AChE in their protonated states (on the 1-DNJ nitrogen atom), the positive charged nitrogen atom is not necessarily involved in cation À p interactions with the aromatic residues of the enzyme 31,33 .
Thus far, five papers have been published, which demonstrate the potential of iminosugars as ChE inhibitors [29][30][31][32][33] . One entry of ChE inhibition by iminosugars that remains to be studied is whether iminosugars can achieve enantioselective ChE inhibition. Thus, in this paper, we present the synthesis of three pairs of optically pure iminosugar-tacrine heterodimer enantiomers, namely, 9a and 9b, 10a and 10b, and 11a and 11b (Scheme 1) and the evaluation of their performance as ChE inhibitors. The study also includes docking studies of the heterodimers to predict interaction with AChE and BuChE. Naturally occurring 1,4dideoxy-1,4-imino-D-arabinitol (DAB) (12a) constitutes the iminosugar moiety in 9a, 10a, and 11a, whereas non-natural 1,4-dideoxy-1,4imino-L-arabinitol (LAB) (12b) is the iminosugar moiety in 9b, 10b, and 11b. Because both iminosugars 21,22 , and heterodimers containing a tacrine moiety 34 have been found to inhibit the growth of cancer cells, we also report the antiproliferative screening of 11a and 11b against a panel of six cancer cell lines.

General procedures
Dichloromethane (DCM), methanol (MeOH), acetone, dimethyl sulfoxide (DMSO) and dimethylformamide (DMF) were dried over 4 Å   Reactions performed at room temperature (rt) refer to the temperature range of 20 to 22 C. TLC analyses were performed on Merck silica gel 60 F 254 plates using UV light (k ¼ 254 nm) for detection. Silica gel NORMASIL 60 V R 40-63 mm was used for silica flash columns. A Bruker Avance NMR spectrometer was used to record 1 H-NMR spectra (400.13 MHz) and 13

Synthetic protocols
General procedure for the preparation of compounds 20a-22a and 20b-22b. A mixture of 19b (1 equiv., 0.04 M for synthesis of 20b, 21b, and 22b) or 19a (1 equiv., 0.07 M for synthesis of 20a, 0.04 M for synthesis of 21a, and 0.05 M for synthesis of 22a), azide 13 (0.98 equiv.), and copper(II) sulphate pentahydrate (0.30 equiv.) in anhydrous DMF in an aluminium foil covered round bottom flask was degassed and introduced an argon atmosphere before the addition of sodium ascorbate (0.60 equiv.). After addition, the mixture was kept stirring for 48 h at rt. The solvent was then removed under reduced pressure and the residue obtained was purified by silica gel column chromatography (See Supplementary Material for details).
General procedure for the preparation of compounds 9a-11a and 9b-11b. To a mixture of 20a-22a (0.02 M, 1 equiv.) or 20b-22b (0.02 M, 1 equiv.) in anhydrous CH 2 Cl 2 under an argon atmosphere at À78 C was slowly added BCl 3 (1 M in heptane, 15 equiv.). After addition, the mixture was kept stirring at À78 C for 2 h and then at 0 C overnight. The volatiles were then removed under reduced pressure and the concentrate underwent purification by gradient silica gel chromatography (MeCN/H 2 O/NH 4 OH 190:10:1 ! 180:20:1) (column 1). The corresponding HCl salt was dissolved in MeOH (2 ml) and NH 4 OH (0.5 ml) and kept stirring for 48 h. The solvent was removed under reduced pressure and the resulting residue was purified by gradient silica gel chromatography (column 2) (the solvent gradient for column 2 for each single experiment is specified in the Supplementary Material).

Inhibition assays
Measuring of the inhibition activity of compounds 9a-11a and 9b-11b against cholinesterases (AChE from Electrophorus electricus and BuChE from equine serum) was accomplished following minor modifications of the Ellman assay 35 , as reported previously 36  fit) implemented in GraphPad Prism 8.01 software; such parameters were in turn used for calculating the inhibition constants of the mixed inhibitors using the following equations: Antiproliferative activity assays For the antiproliferative tests, we applied our implementation of the National Cancer Institute (NCI) screening protocol 39
The synthesis of 9b, 10b, and 11b were performed in the same way as for 9a, 10a, and 11a by replacing L-xylose (14a) with D-xylose (14b) in the first step (Scheme 3).

ChE inhibitory testing
The minimum inhibitory concentrations of the enantiomeric pairs 9a and 9b, 10a and 10b, and 11a and 11b required to reach 50% inhibition (IC 50 ) of eeAChE and eqBuChE are presented in Table 1. A minor modification of the Ellman assay was used in order to measure the IC 50 values 35 . The test series included (-)-galantamine (2a) and tacrine as positive references.
Both series of stereoisomers 9a-11a (incorporating a DAB moiety) and 9b-11b (incorporating a LAB moiety) displayed IC 50 values from the submicromolar concentration range down to the nanomolar concentration range for the inhibition of eeAChE and eqBuChE. The only exception was 9b, which exhibits a IC 50 value of 1480 nM for the inhibition of eeAChE. Thereby, 9b was the only compound in the testing series that is a less potent AChE inhibitor than (-)-galantamine, which is in in current use against AD 47 .
No obvious enantioselectivity of eeAChE and eqBuChE was observed for the three pairs of enantiomeric inhibitors included in this study. In addition, no preferential inhibitory activity trend was found for the enantiomers incorporating a DAB or LAB moiety. For instance, 9a is a ca. 4-fold more potent eeAChE inhibitor than its enantiomer 9b, whereas 10b is a ca. 4-fold more potent eeAChE inhibitor than its enantiomer 10a. For the enantiomeric pair 11a and 11b, we observed essentially equal eeAChE inhibitory activities. These observations indicate that the impact on the eeAChE inhibitory potency of our heterodimers by switching between a DAB and LAB moiety is small compared to the contribution from the tacrine ring.
The inhibition modes of eeAChE and eqBuChE by heterodimer 11b were investigated by using the Cornish-Bowden method, that is, by creating two plots (1/V vs.
[I]) for the inhibition of both enzymes (Figure 3). The two plots for the inhibition of each enzyme included a point of intersection at different [I]coordinates, which implies that 11b is a mixed inhibitor of both enzymes 47 . The competitive inhibition constant, K i , and uncompetitive inhibition constant, aK i , for eeAChE by 11b is 19.0 ± 1.8 nM and 21.9 ± 7.2 nM, respectively. The inhibition constants of eqBuChE are K i ¼10.0 ± 2.7 nM and aK i ¼ 14.3 ± 3.3 nM. The mixed inhibition modes of eeAChE and eqBuChE by 11b were interpreted to indicate that 11b behaves as a dual binding site inhibitor of both enzymes; it is tempting to think that 11b binds simultaneously to the active site and PAS of both eeAChE and eqBuChE. However, in this context it is worth mentioning that the architecture of PAS in the two enzymes is different as it is richer on aromatic amino acid residues in eeAChE 48,49 , which allow formation of p-p interactions and cation-p interactions with ligands 50 .

Modelling studies
The preferred binding poses for enantiomers 11a and 11b in recombinant human acetylcholinesterase (rhAChE) are presented in Figure 4 and Figure 5, respectively, whereas the preferred binding poses for the enantiomeric pairs 9a and 9b, and 10a and 10b are presented in Figure SI2 and Figure SI3, respectively. A common trend for all energetically preferred binding poses is that the tacrine moiety and the iminosugar moiety bind to the active site and PAS, respectively. Such preferred binding pose is not very surprising given that X-ray analysis has shown that tacrine is bound to the active site of AChE 51 .
Hydrogen bonding interactions between one of the hydroxyl groups of the iminosugar moiety and Ser293 in rhAChE are observed in both 11a ( Figure 4) and 11b ( Figure 5). Interestingly, the protonated imino group of 11a showed another hydrogen bond with the same Ser293, a feature not observed in 11b. This helps explain the lower binding energy for 11a (À10.45 kcal/mol) compared to its antipode 11b (À9.92 kcal/mol) ( Table 2). Slight differences in binding energies were also observed between enantiomers 9a and 9b (À9.25 kcal/mol for 9a vs. À9.05 kcal/mol for 9b) and between 10a and 10b (À9.52 kcal/mol for 10a vs. À8.91 kcal/mol for 10b) when they are bound to rhAChE. The  hydroxyl groups of 9a showed interactions with Tyr341 and Arg296 meanwhile there is an arene cation interaction between one of the hydroxyl groups in 9b and Trp286 ( Figure SI2). Hydrogen bond interaction between Ser293 and the iminosugar moiety is observed for 10a but is lacking in its antipode 10b ( Figure SI3). We found that our measured IC 50 values (Table 1) for the inhibition of eeAChE by 11a and 11b are in agreement with the calculated binding energies (Table 2), which predict 11a and 11b to possess the highest affinity for the enzyme. However, IC 50 is not a true measure of binding affinity of a ligand to an enzyme 52 , which explains why the calculated binding energies in Table 2 fail in predicting the relative IC 50 values for the inhibition of eeAChE by the heterodimers (9a-11a and 9b-11b) in our series.
The most energetically favourable binding poses of enantiomers 11a and 11b to human butyrylcholinesterase (hBuChE) are presented in Figure 6 and Figure 7, respectively. The preferred binding poses of the enantiomeric pairs 9a and 9b, and 10a and 10b to the same enzyme are presented in Figures SI5 and SI6, respectively. The number of CH 2 -groups between the tacrine ring and 1,2,3-triazole ring appears to control whether the iminosugar moiety is bound to the active site or PAS. In fact, the tacrine ring of 9a, 9b, 11a, and 11b is accommodated in the active site whereas their iminosugar moiety is bound to PAS. For heterodimers 10a and 10b the binding scenarios are different, as the tacrine ring is accommodated in PAS and the iminosugar moiety in the active site. As for the inhibition of eeAChE, even though the calculated binding energies in Table 2 predict 11a and 11b to be the most potent BuChE inhibitors, they fail in predicting the relative IC 50 values for the whole testing series.

Antiproliferative activity
Antiproliferative activity of our heterodimers was investigated for six cancer cell lines including A549, HBL-100, HeLa, SW1573, T-47D and WiDr. The inhibition of cancer cell growth by each heterodimer is expressed in concentration of heterodimer required to lower the cell growth by 50% (GI 50 ). The antiproliferative activity of a compound is only significant when GI 50 ˂ 100 lM. The measured GI 50 values demonstrated that those heterodimers with two CH 2 -groups (9a and 9b) and three CH 2 -groups (10a and 10b) between the tacrine and 1,2,3-triazole rings display no significant antiproliferative activity (GI 50 > 100 lM). 11a and 11b on the other hand that contain six CH 2 -groups between the tacrine and 1,2,3-triazole rings display GI 50 values below 100 lM for the inhibition of A549 cancer cell growth (Table 3). In addition, 11a and 11b display weak but significant inhibition of cell growth of HeLa and SW1573 cancer cells, respectively.

Conclusions
In contrast to the enantiomeric pairs 1a and 1b of huperzine, 2a and 2b of galantamine, and 3a and 3b of physostigmine, our enantiomeric pairs 9a and 9b, 10a and 10b, and 11a and 11b of iminosugar-tacrine heterodimers displayed low enantioselectivity (˂4) for the inhibition of eeAChE and eqBuChE. The following three observations: (1) 9a is a ca. 3.5-fold stronger eeAChE inhibitor than 9b, (2) 10a is a ca. 3.5-fold less potent eeAChE inhibitor than 10b, and (3) 11a and 11b are essentially equipotent eeAChE inhibitors, show that eeAChE exhibits no consequent preference for any of the enantiomeric heterodimers, which include a DAB or LAB moiety. These observations can either be interpreted as the tacrine moiety contributes much more to the eeAChE inhibitory potencies than the DAB or LAB moieties or that the LAB and DAB moieties have similar contribution to the inhibition potencies when they are connected to a tacrine ring. However, the latter interpretation is to some extent contradicted by the modelling studies, which show that the DAB and LAB moieties display different interaction modes with the enzymes. Like in our earlier studies when we connected an iminosugar to a tacrine ring to obtain ChE inhibitors of type 7 in Figure 2 32 , heterodimers 11a and 11b with the longest linkers exhibited the highest inhibition potencies. From modelling studies for the binding to BuChE, it appeared that the length of the linker between the tacrine ring and DAB or LAB moiety controls whether the tacrine ring binds to the active site of PAS. On the other hand, because the trend of the measured IC 50 values do not parallel the calculated binding energies, it is possible that the title compounds are not bound in their most energetically favourable poses when they inhibit the enzymes in our testing series.